The long-term goal of the proposed research is a detailed structural and mechanistic understanding of the enormously complex macromolecular machine that controls translation initiation in eukaryotes. The proposed work builds on our structural and functional studies of mRNA selection by the cap-binding protein, eukaryotic initiation factor 4E or eIF4E. X-ray crystallography will be combined with chemical methods to study the structures and mechanisms of action of an important subset of the remaining translation initiation factors [eIF4G, eIF4A, eIF2, IF2, eIF3, eIF1A, eIF2B, eIF5, the poly (A)-binding protein (PABP), and a PABP-interacting protein]. These translation factors participate in the following biochemical steps: (a) resolving secondary structural features in the 5' untranslated region of the mRNA, (b) delivering the methionyl initiator tRNA to the 40S subunit, (d) recycling the G protein that loads the methionyl initiator tRNA onto the 40S subunit, (d) recruitment of the 40S subunit, the methionyl initiator tRNA and various accessory factors to the 5' untranslated region of cellular mRNAs bearing 5' 7-methyl-G caps, (e) recruitment of the same components to the 5' untranslated region of an uncapped viral RNA, (f) 60S ribosomal subunit joining, and (g) synergy of transcription initiation via mRNA circularization by the PABP. The specific aims of the research are as follows: Specific Aim 1. Determine X-ray structures of the RNA helicase eIF4A, and its complexes with ADP, a non-hydrolyzable ATP analog, and single-stranded RNA. Specific Aim 2. Determine X-ray structures of eIF2, and its complexes with GDP, a non-hydrolyzable GTP analog, and methionyl initiator tRNA. Specific Aim 3. Determine the X-ray structure of eukaryotic IF2. Specific Aim 4. Determine X-ray structures of a C-terminal fragment of eIF4G, and its complex with a picornavirus internal ribosome entry site. Specific Aim 5. Determine X-ray structures of eIF5 and eIF2Bepsilon, and their complexes with eIF2beta. Specific Aim 6. Determine X-ray structures of PABP recognizing poly (A) RNA, and various binary and ternary complexes with a PABP-interacting protein and eIF4G.